A crucial early step in the formation of excitatory synapses is the physical interaction between the developing presynaptic specialization and the postsynaptic dendrite (Jontes et al., 2000; Ziv and Smith, 1996). This step in excitatory synapse development is thought to be mediated by cell surface membrane proteins expressed by the developing axon and dendrite and appears to be independent of the release of the excitatory neurotransmitter glutamate (reviewed in Dalva et al., 2007). Subsequent steps in excitatory synapse development require the release of glutamate at the developing excitatory synapse. For example, glutamate binding to N-methyl-D-aspartate (NMDA) receptors on the postsynaptic neuron stimulates the maturation of dendritic spines, protrusions on dendrites that are the major sites of excitatory synapses in the brain (Kopec et al., 2006; Matsuzaki et al., 2004). In addition, neuronal activity promotes the use-dependent strengthening of some excitatory synapses and the elimination of many weaker excitatory synapses (reviewed in Greer and Greenberg, 2008).
Several recent studies have revealed an important role for Ephrin cell surface-associated ligands and Eph receptor tyrosine kinases in the early cell-cell contact phase that is critical for excitatory synapse formation (Dalva et al., 2000; Ethell et al., 2001; Henkemeyer et al., 2003; Kayser et al., 2006; Kayser et al., 2008; Lai and Ip, 2009; Murai et al., 2003). Ephs can be divided into two classes, EphA and EphB, based on their ability to bind the ligands EphrinA and EphrinB, respectively (reviewed in Flanagan and Vanderhaeghen, 1998). There are five EphBs (EphB1-4, EphB6), three of which (EphB1-3) are highly expressed in neurons (reviewed in Flanagan and Vanderhaeghen, 1998). EphBs have been mainly shown to be expressed postsynaptically on the surface of developing dendrites, while their cognate ligands, the EphrinBs, are expressed on both the developing axon and dendrite (Grunwald et al., 2004; Grunwald et al., 2001; Lim et al., 2008). When an EphrinB encounters an EphB on the developing dendrite, EphB becomes autophosphorylated, thus increasing its catalytic kinase activity (reviewed in Flanagan and Vanderhaeghen, 1998). This then leads to a cascade of signaling events culminating in actin cytoskeleton remodeling that is critical for dendritic spine and excitatory synapse development (reviewed in Klein, 2009). Consistent with a role for EphBs in excitatory synapse development, EphB1/EphB2/EphB3 triple knockout mice have fewer mature excitatory synapses in vivo in the cortex, and fewer dendritic spines on CA1 and CA3 pyramidal neurons in the hippocampus (Henkemeyer et al., 2003; Kayser et al., 2006). In addition, the disruption of EphB function postsynaptically in dissociated hippocampal neurons leads to defects in spine morphogenesis and a decrease in excitatory synapse number (Ethell et al., 2001; Kayser et al., 2006). Conversely, activation of EphBs in cultured mouse hippocampal neurons leads to an increase in both the number of mature dendritic spines and functional excitatory synapses (Henkemeyer et al., 2003; Penzes et al., 2003; Tolias et al., 2005). Taken together, these findings indicate that EphBs are positive regulators of excitatory synapse development.
EphrinB binding to EphB triggers the recruitment of the NMDA sub-type of glutamate receptors, a calcium-permeable glutamate receptor that has been implicated in the initiation of neuronal activity-dependent synaptic development, excitotoxicity, and neuronal plasticity (Dalva et al., 2000; Takasu et al., 2002). In addition, EphB activation facilitates the recruitment of postsynaptic proteins, including the scaffold protein PSD-95 and the AMPA subtype of glutamate receptors that are critical for postsynaptic development and function (Cabo et al., 2006; Contractor et al., 2002; Henkemeyer et al., 2003; Kayser et al., 2006). The molecular mechanisms by which EphB orchestrates these steps in postsynaptic development are thought to involve EphB-PDZ interactions and the EphB-catalyzed phosphorylation/activation of several guanine nucleotide exchange factors (e.g., Tiam1, Kalirin, and Intersectin) which regulate the Rho family of small G proteins, including Rac1 and Cdc42 (Kayser et al., 2006; Klein, 2009). EphB tyrosine phosphorylation of the RacGEFs Tiam1 and Kalirin7 has been shown to correlate with Rac1 activation and the subsequent activation of p21-activated kinase (PAK) (Penzes et al., 2003, Tolias et al., 2005, Tolias et al., 2007). These events promote changes in the actin cytoskeleton that enhance filopodial motility on the developing dendrite (Kayser et al., 2008). This may then result in an increase in the likelihood that dendritic filopodia will encounter a presynaptic partner and form a synapse. EphB activation also leads to changes in the actin cytoskeleton that are critical for spine morphogenesis and the recruitment of essential postsynaptic components such as NMDA receptors, AMPA receptors, and PSD-95 to the dendritic spine (Dalva et al., 2000; Kayser et al., 2006; Klein, 2009; Penzes et al., 2003). Inhibition of Rac1 or PAK activity blocks the ability of activated EphBs to promote excitatory synapse development, in part through blocking EphB-mediated changes in filopodia movement and possibly by disrupting the recruitment of key proteins to the postsynaptic region (reviewed in Dalva et al., 2007)
While there has been considerable progress in characterizing the mechanisms by which EphBs promote excitatory synapse development, it is not known if there are EphB-associated factors that restrict the timing and extent of excitatory synapse development.
Accordingly, there is a need in the art for methods of determining activators of excitatory synapse formation that can act on EphBs and/or EphB associated factors.